Pulmonary Vasodilation by Sildenafil in Acute Pulmonary Embolism—A Randomized Explorative Trial

Background: To investigate if acute pulmonary vasodilation by sildenafil improves right ventricular function in patients with acute intermediate-high risk pulmonary embolism (PE). Methods: Patients with PE were randomized to a single oral dose of sildenafil 50mg (n=10) or placebo (n=10) as add-on to conventional therapy. Right ventricular function was evaluated immediately before and shortly after (0.5-1.5h) randomization by right heart catheterization (RHC), trans-thoracic echocardiography (TTE), and cardiac magnetic resonance (CMR). The primary efficacy endpoint was cardiac index measured by CMR. Results: Patients had acute intermediate-high risk PE verified by computed tomography pulmonary angiography, systolic blood pressure of 135 ± 18 (mean ± SD) mmHg, increased right ventricular/left ventricular ratio 1.1 ± 0.09 and increased troponin T 167 ± 144 ng/L. Sildenafil treatment did not improve cardiac index compared to baseline (0.02 ± 0.36 l/min/m2, p=0.89) and neither did placebo (0.00 ± 0.34 l/min/m2, p=0.97). Conclusion: A single oral dose of 50 mg sildenafil did not improve cardiac index but lowered systemic blood pressure in patients with acute intermediate-high risk PE.

The ultimate cause of death in PE is right heart failure due to the acute increase in afterload.(7) This is not only due to mechanical obstruction of the proximal pulmonary arteries but also to active pulmonary vasoconstriction mediated by hypoxemia, a neurogenic reflex and the release of vasoconstrictors from activated lung cells, endothelial cells, platelets, and leukocytes. (8-11) As current therapies mainly focus on removing the mechanical obstruction there may be a potential for pulmonary vasodilators as add-on to current treatment strategies. Inhaled nitric oxide (NO) has shown promising results in case reports(12) and the treatment was found to be safe in a phase 1 clinical trial.(13)A recent randomized clinical trial failed at meeting its primary endpoint of normalization of echocardiogram and troponins, but there was improvement in RV function in PE patients treated with NO. (14) Administration of NO is by inhalation trough a nasal cannula or via mechanical ventilation and long-term treatment is not feasible. Pharmacological stimulation of the NO vasodilatory pathway by the oral route may be an attractive alternative to NO therapy.
NO exerts its vasodilator action by up-regulating intracellular cyclic guanosine mono phosphate (cGMP) in the smooth muscle cells of the pulmonary vasculature. cGMP is converted to the inactive 5'GMP by phosphodiesterase type 5 (PDE 5 ). Hence, another strategy for pulmonary vasodilation is inhibition of the breakdown of cGMP by sildenafil, a specific PDE 5 inhibitor widely used in the treatment of pulmonary arterial hypertension. Supporting the biologic rationale for the use of sildenafil in the treatment of PE is a series of pre-clinical studies and few case reports of its efficacy. (15)(16)(17)(18) Furthermore, the ease of use and the low cost makes sildenafil an attractive therapy as addon to conventional therapy if it proves to be effective for the treatment of PE. The aim of this trial was to test the acute effects on right ventricular function of pulmonary vasodilation by sildenafil administered as a single 50 mg oral dose as add-on to conventional therapy in patients with acute intermediate-high risk PE.

Patients
Patients with acute pulmonary embolism confirmed by contrast enhanced computed tomography (CT) were eligible for inclusion if they had symptom duration of less than 14 days, were older than 18 years and had a right ventricular/left ventricular ratio (RV/LV) >1 measured by trans-thoracic echocardiography (TTE, 1 cm above the atrio-ventricular valves in the four-chamber view at enddiastole). Patients were excluded if they were pregnant, had cardiac arrest that required cardiopulmonary resuscitation, a life expectancy <120 days, systolic blood pressure <90 mmHg, metal implants, obesity or claustrophobia that excluded the patient from cardiac magnetic resonance (CMR), altered mental status making the patient unable to provide informed consent, recent use of drugs with influence on the NO-cGMP pathway, known or suspected chronic thromboembolic pulmonary hypertension, inability to perform study protocol < 72 hours after conventional PE treatment was instituted or active bleeding after thrombolysis. Patients were screened for inclusion at hospitals in the Central Region of Denmark

Protocol
The study was designed as single centre, prospective, randomized, placebo-controlled, double-blind, explorative trial. If eligible for inclusion, the patient was transferred to Aarhus University Hospital to complete the study protocol. To characterize the patients, RV/LV ratio was calculated from the CT scan and by TTE performed at the local institution on the day of diagnosis. Cardiac biomarkers (troponin T/I, N-terminal pro-brain natriuretic peptide (NT-proBNP)), height, weight, respiratory frequency, peripheral saturation, and pulse were measured at the local institution on the day of screening and by the study site on the day of inclusion. A detailed medical history was collected including duration of symptoms, known active cancer, recent surgery/trauma, chronic obstructive pulmonary disease, diabetes mellitus, oestrogen therapy, and/or immobilisation.
Detailed protocols for TTE, CMR and RHC are available in the supplemental material. Study drugs (sildenafil or placebo) were prepared in quickly absorbable gelatinous caps by the institutional pharmacy to blind both patients and the physicians to treatment. Patients were randomized in blocks of 10 with sequentially randomly numbered containers. The randomization key was disclosed by the institutional pharmacy after data analysis was complete. All analyses were performed with the observer blinded to treatment. The study was performed and data collected at Aarhus University Hospital, Denmark. The study adheres to CONSORT guidelines.

Statistics
Sample size was calculated to be 10 patients in each treatment group. The calculation was based on the assumptions of a power of 0.9 and α of 0.05 to detect a paired difference of 29 mL/kg/min change in cardiac index with standard deviation of the difference to be 25 mL. Normality testing was performed with the Shapiro-Wilk test. Within-group comparison of continuous data was performed using the two-sided paired t-test. Between-group continuous data and baseline patient characteristics were compared using the two-sided unpaired Students t-test as an explorative analysis. Two-way ANOVA testing was performed to test differences in systemic blood pressure between groups. If data was not normally distributed, non-parametric tests were used (Wilcoxon matched pairs signed-rank or rank sum test) accordingly. All data are presented as mean ± standard deviation (SD). P £ 0.05 was considered statistically significant.

Patients
We enrolled 21 patients from the 1 st of march 2015 through 10 th of September 2017 and randomly assigned 20 patients to a single 50 mg dose of sildenafil (n=10) or placebo (n=10). All patients randomized to treatment had acute PE verified by CT pulmonary angiography, increased RV/LV ratio measured on CT and TTE and increased cardiac biomarkers measured by troponin T/I and/or NT-proBNP. More patients had previous deep venous thromboses in the placebo group. Other baseline characteristics were similar in both groups (Table 1). Time from hospital admission and initiation of conventional therapy to study inclusion were 2.3 ± 0.7 days. Mean CI for all patients included was 2.3 ± 0.6 l/min/m 2 . CMR was performed in all patients. Inclusion rate was low initially as several patients wanted to avoid right heart catheterization (RHC). To improve inclusion rate, RHC was made optional 4 months after initiation of the study. RHC was performed in 16 patients (sildenafil n=8 and placebo n=8). TTE were performed in all patients, but the results from 2 patients were excluded before unblinding due to poor image quality (placebo n=2). One patient did not have RV/LV >1on TTE on the day of inclusion and was excluded as screening failure before randomization.

Primary and explorative endpoints for right ventricular function
Primary and explorative endpoints are summarized in figure 2A  sildenafil significantly reduced end-diastolic and end-systolic RV volume. mPCWP increased with placebo but was unaltered by sildenafil. PAAT was shortened after placebo treatment but was unaltered by sildenafil. Other exploratory endpoints were unaltered both by sildenafil and placebo.

Safety
None of the patients reported adverse events. Blood pressure was lower in sildenafil treated patients compared to placebo (figure 2B). The maximal drop in blood pressure was observed 120 min after administration of study drug (sildenafil: -19 ± 10, placebo: 0 ± 9 mmHg, p=0.0007) to a MAP of 91 ± We used several independent methods to evaluate RV function. Previous and ongoing studies on a similar topic have used RV/LV ratio measured by echo as a primary endpoint. 21,22 By the use of a more elaborate RV evaluation in this study we could potentially measure subtle hemodynamic changes even with a low number of patients. In the exploratory endpoints we did find a reduction in EDV and ESV. With an unaltered PVR these changes could simply be secondary to the observed lower systemic blood pressure which emphasizes the accuracy of the hemodynamic evaluation.
We chose to test the acute effects of pulmonary vasodilation after administration of a single 50 mg dose of sildenafil as this design has previously proven to be effective in PAH patients(22) and in preclinical studies of PE. (18) The observation of a lowered MAP at several independent timepoints after administration similar to previous observations(22) confirm that sildenafil was administered in an effective dose and that the effect was evaluated at relevant timepoints after administration.
The magnitude of MAP lowering together with the observation that none of the patients reported adverse events suggest that sildenafil was safe in our patients. It should however be emphasized that data is limited and we cannot conclude that sildenafil is generally safe in acute PE based on our data alone.

Limitations
There are some limitations to our study. One is the delay of 2.3 days from initiation of conventional therapy to study inclusion. It has been suggested that vasoconstriction is more pronounced in the early phase after PE and not so much in the later phase.(23,24) Hence the potential effect of pulmonary vasodilation in acute PE would be blunted at study inclusion more than 2.3 days after hospital admission. There are limited data supporting this speculation but it could explain the discrepancy between the positive experimental studies that initiate pulmonary vasodilation shortly after PE and the few, but all neutral clinical studies where onset of treatment is delayed.
Another limitation is the low number of patients included in the trial. Being an exploratory study we choose an elaborate hemodynamic evaluation of the patients to identify possible hemodynamic changes. Post-hoc power calculation revealed that 3924 patients should be included to show a significant difference with a power of 0.8 and α of 0.05 to detect a paired difference of the observed 17 mL/m 2 /min change in cardiac index using the observed standard deviation of 380 mL/m 2 /min. Looking at Figure 2 however, there is one sildenafil treated patient with a CI of 3.8 L/m 2 /min at baseline and as discussed previously there would be rationale behind excluding patients with a high cardiac output in future trials. If this was done in this trial with a cut-off of CI < 3 L/m 2 /min, a nonsignificant improvement of 112 mL/m 2 /min was observed (p=0.2). Although speculative, this observation could be of use in future trials. A post hoc power calculation for the more widely used RV/LV ratio as primary endpoint using the observed 0.04 change in RV/LV ratio with SD of 0.16 reveals that 128 patients should be included to show an effect in the treatment arm with a power of 0.8 and α of 0.05 suggesting that RV/LV ratio may be a more robust primary endpoint in future trials.
The calculations above discuss statistical effect. Whether or not these small improvements in hemodynamics would translate if translate into a clinical effect is unknown.
The limitations in this trial should be taken into consideration in the interpretiation of the data. There is substantial experimental evidence in a wide range of PE models that pulmonary vasodilation improves hemodynamics in acute PE(10) but still, there are only a very limited number of small clinical trials. Therefore it would still be relevant to explore the clinical potential of pulmonary vasodilation in acute PE. Patient selection with regards to high RV/LV ratio, low CI and timing will probably be key to success in future trials.

Conclusion
In conclusion we found that a single dose of sildenafil 50 mg lowered systemic blood pressure but did

Funding
The study was investigator initiated and driven. The study was funded by the Novo Nordisk Foundation (NNF16OC0023244). NNF had no role in designing the study, data collection, data analysis, data interpretation or writing of the report.    Figure 1 Study design. CMR cardiac magnetic resonance imaging. TTE trans-thoracic echocardiography. RCH right heart catheterization. R randomisation sildenafil 50 mg or placebo. * measurement of blood pressure.